A Structures for Lossless Ion Manipulations (SLIM) … · A Structures for Lossless Ion Manipulations (SLIM) ... P., Aebersold, R.: Selected reaction monitoring-based prote- ... A

B American Society for Mass Spectrometry, 2016DOI: 10.1007/s13361-016-1397-x

J. Am. Soc. Mass Spectrom. (2016) 27:1285Y1288

A Structures for Lossless Ion Manipulations (SLIM) Modulefor Collision Induced Dissociation

Ian K. Webb, Sandilya V. B. Garimella, Randolph V. Norheim, Erin S. Baker,Yehia M. Ibrahim, Richard D. SmithBiological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 3335Innovation Ave. (K8-98), P.O. Box 999, Richland, WA 99352, USA

Abstract. A collision induced dissociation (CID) structure for lossless ion manipula-tions (SLIM) module is introduced and coupled to a quadrupole time-of-flight (QTOF)mass spectrometer. The SLIM CID module was mounted after an ion mobility (IM)drift tube to enable IM/CID/MS studies. The efficiency of CIDwas studied by using themodel peptide leucine enkephalin. CID efficiencies (62%) compared favorably withother beam-type CID methods. Additionally, the SLIM CID module was used tofragment a mixture of nine peptides after IM separation. This work also representsthe first application of SLIM in the 0.3 to 0.5 Torr pressure regime, an order ofmagnitude lower in pressure than previously studied.

Keywords: Collision induced dissociation, Ion mobility spectrometry, rf Confinement, Ion optics, Peptide frag-mentation, Manipulation, Conveyor

Received: 1 February 2016/Revised: 15 March 2016/Accepted: 23 March 2016/Published Online: 20 April 2016

Introduction

C ollision induced dissociation (CID) is ubiquitous in massspectrometry (MS). Since the advent of widely used

Bsoft^ ionization methods (viz. electrospray ionization (ESI)and matrix-assisted laser desorption ionization (MALDI) [1,2]), CID has been invaluable for proteomics identification [3–8] and quantitation [9–11]. An important CID figure of merit isthe CID efficiency (ECID; Equation 1)

ECID ¼X

Iproduct� �

100%ð ÞI0

¼ EFragmentation

� �ECollectionð Þ 100%ð Þ ð1Þ

where Iproduct is product ion intensity and I0 is the initialprecursor ion intensity. CID efficiency can also be calculated

as the product of fragmentation efficiency (Equation 2) andcollection efficiency (Equation 3).

EFragmentation ¼X

Iproduct� �

100%ð ÞX

Iproducts þ Iprecursorð2Þ

ECollection ¼X

Iproducts þ Iprecursor� �

100%ð ÞI0

ð3Þ

where Iprecursor is remaining precursor ion intensity.Previously, fragmentation techniques, such as photodisso-

ciation [12], surface-induced dissociation [13, 14], andcollision-induced dissociation [15–22] have been coupled toIMS for mobility-separated fragmentation of precursor ions.Fragment ions will retain the arrival ions of their respectiveprecursors, provided the fragmentation occurs after the IMseparation. In addition, recent reports have also demonstratedIM-selection for action spectroscopy [23, 24]. In this work,we introduce a new structure for lossless ion manipulations(SLIM) CID module for CID/MS. SLIM devices have beenpreviously demonstrated for ion mobility (IM) separations[25–28], mobility-based ion selection [29], and ion trapping[30]. In this study, we demonstrate that SLIM is adaptable

Electronic supplementary material The online version of this article (doi:10.1007/s13361-016-1397-x) contains supplementary material, which is availableto authorized users.

Correspondence to: Richard D. Smith; e-mail: [email protected]

RESEARCH ARTICLE

(and highly suitable) to applications outside of IM. We alsoshow that SLIM devices are not limited to ~4 Torr and canprovide effective ion transmission at lower pressures. TheSLIM CID module was used to dissociate ions after an IMstage, providing fragmentation precursor peptides after a mo-bility separation. In this work, we used the well-studied ESIthermometer ion, leucine enkephalin [31], as well as a mix-ture of nine peptides, to evaluate the effectiveness of theSLIM CID module.

ExperimentalLeucine enkephalin was prepared in a 1 μMsolution of 50/50/1(vol/vol/vol) water/methanol/acetic acid. An equimolar 1 μM/each solution of nine peptides (angiotensin I and II, bradykinin,fibrinopeptide A, kemptide, melittin, neurotensin, renin sub-strate tetradecapeptide, and substance P) was also prepared in50/50/1 water/methanol/acetic acid. Water was purified by aBa rn s t e ad Nanopu r e s e t t o 18 MΩ r e s i s t i v i t y(ThermoScientific, Waltham, MA, USA); peptides were pur-chased from Sigma-Aldrich (St. Louis, MO, USA), and meth-anol and acetic acid were purchased from Fisher Scientific(Pittsburgh, PA, USA). Solutions were infused at 300 nL/minfrom a chemically etched emitter nanoelectrospray source [32]into a home-built IM/MS [21, 22, 33]. The design of the SLIMmodule, shown in Figure 1, was based upon previous designsused for ion mobility separations [25] and other manipulations[30, 34]. Briefly, ions are confined laterally using DC from‘guard’ electrodes and vertically by pseudopotentials generatedby rf applied in opposite phase to each adjacent electrode. TheDC/rf electrodes have a superimposed DC gradient appliedacross a section of electrodes such that ions will experience aconstant electric field and traverse the device from high to lowDC potentials (left to right in Figure 1). Two SLIM surfacesfabricated from PCBs are then mounted parallel to each other.The DC-only guards are black in Figure 1, and electrodes withboth DC and rf are red. There are three independently control-lable DC regions on the device. The first two regions (gradients1 and 2) each span 11 DC-only guard electrodes, and the final

region contains a planar quadrupole-like geometry (exit region)to focus ions into the center of the device for entrance into themass spectrometer (Agilent 6538 QTOFMSwith a 1.5 m flighttube; Agilent Technologies, Santa Clara, CA, USA).Therefore, higher electric fields suitable for CID can be appliedacross two regions: between the first two gradients and betweenthe second gradient and quadrupole-like region. The DC fieldsin the gradients were restricted to a maximum of 15–16 V/cm,so that fragmentation was minimized when CID fields were notapplied. The guards in the first two regions were biased 10 VDC

higher than the neighboring rf/DC electrodes. All three elec-trodes in the exit region were biased to the same VDC. For IM/CID/MS, ions were accumulated in the ion funnel trap [35–37]and released into the drift tube (4 Torr, 16 V/cm constant field)in 488 μs pulses. The drift tube was followed by a rear ionfunnel with a conductance limiting orifice, a short rf-onlytransmission quad, another conductance limiting orifice, andthe SLIM CID module. Therefore, the pressure in the SLIMregion could be varied without affecting the drift tube pressure.

ResultsLeucine enkephalin was chosen as a model peptide for fragmen-tation as the fragmentation patterns are widely documented andunderstood [31]. Figure 2 shows representative spectra of proton-ated leucine enkephalin. The DC voltage in Figure 2a betweenthe end of gradient 2 and the exit region (VCID) was 0 V.Applying a VCID of 30 V (Figure 2b) results in extensive frag-mentation, including cleavage of all peptide bonds (y4, b2, b3, andb4). The CID efficiency was 62%, comparing favorably to effi-ciencies in a triple-quadrupole and for 200 mTorr in a segmentedquadrupole CID (36% in both cases) [21], and dipolar resonantexcitation CID ofmethionine enkephalin at 80mTorr (44%) [22].

Next, the CID efficiencies from applying VCID between gra-dient 1/gradient 2 and gradient 2/exit region were measured(Supplementary Figure 1). VCID was increased for each caseuntil the fragmentation efficiency remained roughly constant(Equation 2). The maximum CID efficiency from gradient 2/exit region CID was 62% (30 VCID). The maximum efficiencywhen VCID was applied between gradients 1 and 2 was 50%(20 VCID). The fragmentation efficiency for VCID between gra-dient 2 and the exit region was 80% and between gradient 1 andgradient 2 was 84%. Therefore, the increase in CID efficiency forapplication of VCID in between gradient 2 and the exit region wasdue to increased collection efficiency (77% versus 60%).Although both methods are equally efficient with the applicationof 20 VCID, the collection efficiency was 60% between gradients1 and 2 and 81%between gradient 2 and the exit region. Changesin collection efficiency are likely due to stronger ion focusing ofproduct ions in the quadrupolar region than the rf/DC region,where ions move in closer proximity to surfaces [26, 27].

Figure 3 shows two nested IM/MS spectra of a nine peptidemix. Figure 3a was taken with 0 VCID, and Figure 3b was takenwith a 45VCID potential between gradient 2 and the exit region.After the application of CID, characteristic dissociation

Figure 1. Layout of electrodes on one of the two planar SLIMsurfaces. Black (guard) electrodes are DC-only, and red elec-trodes are rf/DC. The DC was divided into two separate gradi-ents of equal length, followed by an independently controlledDC for the planar pseudo-quadrupole region at the exit, where rfon the guard electrodes is the same phase and the rf on thecentral electrode is 180 out of phase with respect to the guards.Ions traverse the SLIM module from left to right

1286 I. K. Webb et al.: SLIM CID

Bladder^ patterns appear in the nested spectra. The product ionsin the nested spectra appear vertically aligned with the arrivaltime of the precursor. Extensive dissociation was observed,showing the utility of IM/SLIM CID for ‘all-ion’fragmentation.

ConclusionsWe have introduced a CID-capable SLIM module includingtwo CID regions. The most efficient CID was observed whenthe VCID was applied between the second voltage gradient andthe exit region. SLIM CID resulted in extensive fragmentationof the thermometer peptide ion protonated leucine enkephalin.SLIM CID coupled to an IM separation was exemplified withall-ion fragmentation of a mixture of peptides. In the future,SLIM CID will be coupled to high resolution SLIM IM sepa-rations to give higher peak capacities and direct connectivity ofprecursor ions to product ions without requiring mass selec-tions for data-independent analysis experiments. Additionally,this study showed the ability of SLIM devices to transmit ionsat lower pressures than pressures used in previous studies (i.e.,

4 Torr). Present work is ongoing to optimize SLIM CID forhigher pressures for more direct coupling to IM measurementswithout losses of resolving power or sensitivity due to changesin pressure. Once SLIM CID is integrated into existing SLIMmodules, slower heating trapping/longer activation time exper-iments can be performed, which will allow for higher CIDefficiencies.

AcknowledgmentsPortions of this research were supported by the NationalInstitutes of Health (NIH) NIGMS grant 5P41GM103493-13 (R.D.S.), by the Department of Energy, Office of Biolog-ical and Environmental Research Genome Sciences Programunder the Pan-omics project, and the Laboratory DirectedResearch and Development (LDRD, I.K.W. and E.S.B.) pro-gram at the Pacific Northwest National Laboratory. Workwas performed in the Environmental Molecular Science Lab-oratory, a U.S. Department of Energy (DOE) national scien-tific user facility at Pacific Northwest National Laboratory

2030

200

300

400

500

600

700

800

900

1000

1100

Drift Time (ms)

m/z

5000

00

2500

00 0

Intensity (a.u.) 0

400000

800000

Inte

nsity

(a.

u.)

(a)

(b)2030

200

300

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500

600

700

800

900

1000

1100Drift Time (ms)

m/z

2500

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1250

000 0

Intensity (a.u.) 0

100000

200000

Inte

nsity

(a.

u.)

Figure 3. Nested IM/MS spectra of a mix of nine peptides at365 mTorr, 750 kHz, 200 Vp-p rf. (a) 0 VCID, (b) 45 VCID

Figure 2. Representative spectra of protonated leucine en-kephalin at 265 mTorr, 750 kHz, 200 Vp-p rf. (a) VCID =0. (b)VCID=30, CID efficiency=62%

I. K. Webb et al.: SLIM CID 1287

(PNNL) in Richland, WA. PNNL is operated by Battelle forthe DOE under contract DE-AC05-76RL0 1830.

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